Project Summary The long term goal of the active parent award is to establish an efficient system for the culture of human spermatogonial stem cells (SSCs). Availability of safe, efficient strategies to propagate SSCs in culture will help develop new treatment options for male infertility as well as preservation and restoration of fertility in cancer patients undergoing potentially sterilizing treatments. The project uses pigs as an established, accessible animal model phylogenetically and physiologically more similar to humans than rodents. Key findings will be translated to human SSCs. Interactions with the somatic microenvironment in the testis govern germ lineage specification and germ cell differentiation. Modeling this niche environment requires appropriate in vitro systems. Previous studies on human germ cell specification used fetal gonadal tissue to establish key events of germ cell differentiation and to inform induced differentiation of pluripotent stem cells (PSCs) into early germ cells. Differentiation is largely dependent on cell-cell interactions between germ cells and the somatic environment, which was explored by aggregation of PSC-derived germ cells with fetal gonadal somatic cells. Specific to the Notice of Special Interest (NOT-HD-19-011), we build on the organotypic testicular organoids we developed from porcine and human cells in Specific Aim 3 of the parent grant, and existing porcine and human iPSC lines to model the inductive effects of the testicular microenvironment on germ lineage differentiation as an alternative for using fetal gonadal tissue. While PSC-derived germ cells are not suitable for fertility preservation, a better understanding of germ cell-somatic cell interactions in the testis can be translated to culture conditions for more efficient expansion of human SSCs for fertility preservation. We will address one Supplemental Aim: To explore effects of the testicular organoid microenvironment on germ cell differentiation from iPSCs. We will test the hypothesis that the organoid microenvironment will support differentiation of iPSC-derived germ cells. We will study the inductive effect of the organoid microenvironment on germ lineage differentiation of iPSCs, and monitor germ cell differentiation from iPSC-derived germ cells in the organoid niche. Short term experiments in vitro will be complemented by grafting of organoids carrying iPSC-derived germ cells to mouse hosts to observe differentiation in a supportive in vivo environment. So far, testicular organoids have been generated from primary testis cells. To expand the utility of the organoid platform, we will test the hypothesis that functional testicular organoids can be generated from iPSCs. Testicular somatic cells derived by directed differentiation of iPSCs following published protocols will be combined in the microwell system to form testicular organoids. Testis specific organization and functions will be monitored by expression of cell type specific proteins and support of germ cell differentiation. Taken together, our experimental results will provide an integrated system to model testis development and germ cell differentiation without need for fetal gonadal tissue. Insights gained will help expand human SSCs in culture for clinical application to preserve and restore male fertility.